Primer set for multiple detecting Solanaceae viruses and method of multiple detecting Solanaceae viruses using the same

Abstract

The present invention relates to a primer set capable of simultaneously diagnosing major viruses occurring in horticultural crops, such as Broad Bean Wilt Virus, Beet Western Yellow Virus, Tomato Bushy Stunt Virus, Tomato Chlorosis Virus, Pepper Mottle Virus, Pepper Mild Mottle Virus, Cucumber Mosaic Virus, Tomato Spotted Wilt Virus, and Tomato Yellow Leaf Curl Virus, by utilizing a real-time gene amplifier, a composition for markers including the same, a kit, and a diagnostic method using the same.

Description

Technology Field

[0001] The present invention relates to a primer set for multiplex diagnosis of viruses in Solanaceae crops and a multiplex diagnosis method using the same, and more specifically, to a method utilizing a real-time gene amplifier for the broad bean wilt virus, a major virus occurring in horticultural crops ( Broad Bean Wilt Virus ), beet yellowing virus ( Beet Western Yellow Virus ), Tomato Bush Atrophy Virus( Tomato Bushy Stunt Virus ), Tomato chlorosis virus( Tomato chlorosis virus ), chili pepper stain virus( Pepper Mottle Virus ), chili pepper light stain virus( Pepper Mild Mottle Virus ), cucumber mosaic virus( Cucumber Mosaic Virus ), Tomato Spotted Wilt Virus( Tomato Spotted Wilt Virus ), Tomato Yellow Leaf Curl Virus( Tomato Yellow Leaf Curl Virus This invention relates to a primer set capable of simultaneously diagnosing ) and a diagnostic method using the same. Background Technology

[0002] The total greenhouse cultivation area for major domestic fruit and vegetable crops is 47,921 hectares for 10 crops including chili peppers and tomatoes, with a production volume of 2,122,417 tons. Of this, chili peppers, tomatoes, and paprika, which are Solanaceae crops, account for 22.2% of the total cultivation area and 28.7% of the production volume (Agricultural and Forestry Statistics, 2020).

[0003] The cultivation area and production volume of Solanaceae crops in Gangwon-do are 906 ha / 47,733 tons for tomatoes, 812 ha / 26,467 tons for green peppers, and 258 ha / 22,289 tons for paprika. When compared to the national cultivation area, tomatoes account for 16.4% / 13.8%, peppers 18.5% / 14.4%, and paprika 35.1% / 27.2%. These Solanaceae crops are cultivated intensively in most greenhouse facilities, and as open-field land is shrinking due to climate change, they are important crops as a source of high income for farmers.

[0004] There are a total of 19 types of viruses affecting these vegetables, and regarding the viruses causing the most damage, in the cases of 2008 (Kim et al., 2009) and 2009 (Choi et al., 2010), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), Broad bean wilt virus Four types, including 2(BBWV2), were reported.

[0005] Viruses that occurred in Solanaceae crops in Gangwon-do from 2014 to 2018, too Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), Broad bean wilt virus Eight types of viruses, including 2 (BBWV2), have been reported (Won et al., 2018).

[0006] The incidence of such viral diseases is rising every year as the number of vector insects that transmit viruses increases due to the impact of climate change. From 2017 to 2022, a total of 374 complaints regarding disease were received in Gangwon-do, with viruses accounting for 52% of them.

[0007] There are 24 types of viruses diagnosed, including CMV, and the most frequently diagnosed viruses are TSWV, CMV, BBWV, and TYLCV. The reason for the high number of complaints regarding viral disease outbreaks is the lack of effective treatments. Therefore, when a viral disease occurs, active control measures such as controlling insect vectors and removing infected plants are the best approach; however, for effective active control, it is crucial to rapidly diagnose the specific type of virus affecting the field.

[0008] Currently, when viral diseases occur, diagnostic methods include using rapid diagnostic kits or the conventional PCR method. The rapid diagnostic method uses a strip kit to confirm whether a test is positive based on an antigen-antibody reaction against a suspected virus strain. While it has the advantage of being able to confirm results within 10 minutes, it is only possible with a single virus, and the types of viruses that can be diagnosed are also limited.

[0009] Conventional PCR methods such as those described in Patent Document 1 and Patent Document 2 can diagnose various types of viruses, but they require the addition of separate virus primers for diagnosis and take about 5 to 6 hours to verify the results through an electrophoresis process after the PCR is completed. Consequently, there is a concern that by the time the diagnostic results are obtained, the viral disease may have already spread within the packaging.

[0010] Therefore, only by rapidly diagnosing suspected viruses within a short period of time can the spread of the disease be prevented. Prior art literature

[0011] Korean Patent Publication No. 10-2033293 Korean Patent Publication No. 10-2175124 The problem to be solved

[0012] The objective of the present invention is to provide a primer set for multiple diagnosis of viruses in Solanaceae crops.

[0013] Another objective of the present invention is to provide a marker composition for multi-diagnostic of viruses in Solanaceae crops.

[0014] Another objective of the present invention is to provide a kit for multi-diagnostic of viruses in Solanaceae crops.

[0015] Another objective of the present invention is to provide a method for multi-diagnosing viruses in Solanaceae crops. means of solving the problem

[0016] To achieve the above objectives, the present invention comprises: a first primer set consisting of a forward primer of SEQ ID NO. 1, a reverse primer pair of SEQ ID NO. 2, and a probe of SEQ ID NO. 19; a second primer set consisting of a forward primer of SEQ ID NO. 3, a reverse primer pair of SEQ ID NO. 4, and a probe of SEQ ID NO. 20; a third primer set consisting of a forward primer of SEQ ID NO. 5, a reverse primer pair of SEQ ID NO. 6, and a probe of SEQ ID NO. 21; a fourth primer set consisting of a forward primer of SEQ ID NO. 7, a reverse primer pair of SEQ ID NO. 8, and a probe of SEQ ID NO. 22; a fifth primer set consisting of a forward primer of SEQ ID NO. 9, a reverse primer pair of SEQ ID NO. 10, and a probe of SEQ ID NO. 23; a sixth primer set consisting of a forward primer of SEQ ID NO. 11, a reverse primer pair of SEQ ID NO. 12, and a probe of SEQ ID NO. 24; and a seventh primer set consisting of a forward primer of SEQ ID NO. 13, a reverse primer pair of SEQ ID NO. 14, and a probe of SEQ ID NO. 25. A primer set for multiplex diagnosis of viruses in Solanaceae crops is provided, comprising: an eighth primer set consisting of a forward primer of SEQ ID NO. 15, a reverse primer pair of SEQ ID NO. 16, and a probe of SEQ ID NO. 26; and a ninth primer set consisting of a forward primer of SEQ ID NO. 17, a reverse primer pair of SEQ ID NO. 18, and a probe of SEQ ID NO. 27.

[0017] In one embodiment of the present invention, the first primer set may be for diagnosing sideburn wilt virus (BBWV).

[0018] In one embodiment of the present invention, the second primer set may be for diagnosing beet yellowing virus (BWYV).

[0019] In one embodiment of the present invention, the third primer set may be for diagnosing Tomato Bush Sickness Virus (TBSV).

[0020] In one embodiment of the present invention, the fourth primer set may be for diagnosing tomato yellowing virus or tomato chlorosis virus (ToCV).

[0021] In one embodiment of the present invention, the fifth primer set may be for diagnosing pepper spot virus (PepMoV).

[0022] In one embodiment of the present invention, the sixth primer set may be for diagnosing pepper mildew virus (PMMoV).

[0023] In one embodiment of the present invention, the seventh primer set may be for diagnosing cucumber mosaic virus (CMV).

[0024] In one embodiment of the present invention, the eighth primer set may be for diagnosing Tomato Spotted Wilt Virus (TSWV).

[0025] In one embodiment of the present invention, the ninth primer set may be for diagnosing Tomato Yellow Leaf Curl Virus (TYLCV).

[0026] In one embodiment of the present invention, the crop of the Solanaceae family may be one or more selected from chili peppers, paprika, tomatoes, eggplants, potatoes, and bell peppers.

[0027] To achieve the above objectives, the present invention provides a marker composition for multiplex diagnosis of viruses in Solanaceae crops, comprising a primer set for multiplex diagnosis of viruses in Solanaceae crops.

[0028] To achieve the above objectives, the present invention provides a kit for multi-diagnostic of viruses in Solanaceae crops comprising a marker composition for multi-diagnostic of viruses in Solanaceae crops.

[0029] To achieve the above objectives, the present invention provides a method for multiplex diagnosis of viruses in Solanaceae crops, comprising: (a) a step of extracting RNA from a virus-infected plant; (b) a step of performing an amplification reaction using the RNA extracted in step (a) as a template and the marker composition of claim 11; and (c) a step of detecting the amplification product obtained in step (b).

[0030] In one embodiment of the present invention, the amplification in step (b) may utilize a multiplex qRT-PCR method.

[0031] In one embodiment of the present invention, detection of the amplified product of step (c) may be performed using any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof. Effects of the invention

[0032] The present invention enables the rapid detection of diseases caused by nine types of viruses—broad bean wilt virus (BBWV), beet western yellow virus (BWYV), tomato bushy stunt virus (TBSV), tomato chlorosis virus, pepper mottle virus (PepMoV), pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV), and tomato yellow leaf curl virus (TYLCV)—occurring in Solanaceae crops such as peppers, watermelons, and cucumbers, using primer sets and probes. Furthermore, since the time required for detection and diagnosis can be shortened by using a multiplex qRT-PCR method capable of simultaneously detecting up to nine viruses in a single experiment, the invention can be usefully applied to the diagnosis of diseases in Solanaceae crops. It is.

[0033] The 9 viruses are divided into 3 sets of 3 each, and for the convenience of the analyst, the 3 sets are conducted with equal experimental methods and conditions, allowing the analyst to arbitrarily divide the virus sets into 3, 6, or 9 for analysis. Brief explanation of the drawing

[0034] Figures 1a and 1b show the primer set with the best PCR efficiency, in which the Ct value in the amplification curve results is less than 20, no false positive results occur in NTC, and reproducibility is shown in the 2-repetition results. Figures 2a and 2e show each gene region selected to design a set of primers and probes for detecting nine viruses. Figures 3a and 3b show the results of selecting one probe for each virus that has the best PCR efficiency, with a Ct value less than 20 in the amplification curve results and no false positive results in NTC. Figure 4 shows the primers and probes for detecting the virus configured in Figure 5, with 6 primers and 3 probes included per tube. Since the primers and probes must not bind to each other to generate non-specific fluorescence, this is an example of data used to check for the formation of mono-dimers (binding between identical primers), hetero-dimers (binding between different primers), and hairpins (binding within a single primer / probe sequence) using the primer / probe sequences. Figure 5 shows the development of a Multiplex qPCR diagnostic kit in which 9 types of viruses related to Solanaceae crops were divided into 3 types each and arranged into #A, #B, and #C tubes, indicating the types of viruses detected according to the fluorescence of each tube. Figure 6 shows the results of detection based on whether no false positive results occur in NTC, where the Ct value in the amplification curve result is less than 20. Figures 7 to 9 show the results of analyzing the Ct values ​​of the amplification curves after diluting the RNA of 9 types of Solanaceae crop viruses from 10 ng to 100 fg. Specific details for implementing the invention

[0035] A first embodiment of the present invention comprises: a first primer set consisting of a forward primer of SEQ ID NO. 1, a reverse primer pair of SEQ ID NO. 2, and a probe of SEQ ID NO. 19; a second primer set consisting of a forward primer of SEQ ID NO. 3, a reverse primer pair of SEQ ID NO. 4, and a probe of SEQ ID NO. 20; a third primer set consisting of a forward primer of SEQ ID NO. 5, a reverse primer pair of SEQ ID NO. 6, and a probe of SEQ ID NO. 21; a fourth primer set consisting of a forward primer of SEQ ID NO. 7, a reverse primer pair of SEQ ID NO. 8, and a probe of SEQ ID NO. 22; a fifth primer set consisting of a forward primer of SEQ ID NO. 9, a reverse primer pair of SEQ ID NO. 10, and a probe of SEQ ID NO. 23; a sixth primer set consisting of a forward primer of SEQ ID NO. 11, a reverse primer pair of SEQ ID NO. 12, and a probe of SEQ ID NO. 24; and a seventh primer set consisting of a forward primer of SEQ ID NO. 13, a reverse primer pair of SEQ ID NO. 14, and a probe of SEQ ID NO. 25. The present invention relates to a primer set for multiplex diagnosis of viruses in Solanaceae crops, comprising: an 8th primer set consisting of a forward primer of SEQ ID NO. 15, a reverse primer pair of SEQ ID NO. 16, and a probe of SEQ ID NO. 26; and a 9th primer set consisting of a forward primer of SEQ ID NO. 17, a reverse primer pair of SEQ ID NO. 18, and a probe of SEQ ID NO. 27.

[0036] In the present invention, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a template of complementary nucleic acid, and functioning as a starting point for strand replication of the nucleic acid template. The primer can initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

[0037] When designing the primers mentioned above, various constraints apply, such as the ratio of A, G, C, and T content, prevention of primer dimer formation, and prohibition of repeating the same nucleotide sequence three or more times; additionally, regarding single PCR reaction conditions, the amount of template DNA, primer concentration, dNTP concentration, and Mg 2+ Conditions such as the intended use, reaction temperature, and reaction time must be appropriate.

[0038] The above primers may incorporate additional features without changing their basic properties. That is, the nucleic acid sequence may be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more nucleotides with homologues, and modification of nucleotides into uncharged linkages such as phosphonates, phosphotriesters, phosphoromidates, or carbamates, or charged linkages such as phosphorothioates or phosphorodithioates.

[0039] In addition, nucleic acids may have one or more additional covalently bonded residues, such as proteins including nucleases, toxins, antibodies, signal peptides, and poly L-lysine; intercalators such as acridine or psoralen; chelating agents such as metals, radioactive metals, and iron oxide metals; and alkylating agents.

[0040] Additionally, the primer sequences of the present invention may be modified using labels that can provide a detectable signal directly or indirectly. The primers may include labels that can be detected using spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful labels include 32P, fluorescent dyes, electron-concentrating reagents, enzymes (typically used in ELISA), biotin or hapten, and proteins for which antiserum or monoclonal antibodies are available.

[0041] The primers of the present invention may be chemically synthesized using any other widely known method including cloning of a suitable sequence and restriction enzyme digestion and direct chemical synthesis methods such as the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68:90-99), the diethylphosphoramidite method of Beaucage et al. (1981, Tetrahedron Lett. 22: 1859-1862), and the solid support method of U.S. Patent No. 4458066.

[0042] The first primer set is for diagnosing Big Beef Flesh Virus (BBWV), the second primer set is for diagnosing Beetroot Yellow Virus (BWYV), the third primer set is for diagnosing Tomato Bush Sickness Virus (TBSV), the fourth primer set is for diagnosing Tomato Yellow Virus or Tomato Chlorosis Virus (ToCV), the fifth primer set is for diagnosing Pepper Mosaic Virus (PepMoV), the sixth primer set is for diagnosing Pepper Mosaic Virus (PMMoV), the seventh primer set is for diagnosing Cucumber Mosaic Virus (CMV), the eighth primer set is for diagnosing Tomato Spotted Wilt Virus (TSWV), and the ninth primer set may be for diagnosing Tomato Yellow Leaf Curl Virus (TYLCV).

[0043] In one embodiment of the present invention, the crop of the Solanaceae family may be one or more selected from chili peppers, paprika, tomatoes, eggplants, potatoes, and bell peppers, but is not limited thereto.

[0044] A second embodiment of the present invention relates to a marker composition for multiplex diagnosis of viruses in Solanaceae crops, comprising a primer set for multiplex diagnosis of viruses in Solanaceae crops.

[0045] The above composition may further include a reaction amplification mixture, the reaction amplification mixture refers to a reagent necessary for performing an amplification reaction, a heat-stable DNA polymerase, a deoxynucleotide, nuclease-free sterile water, and a solution containing a divalent metal cation, and preferably may include a reaction buffer, a deoxynucleotide, and a DNA polymerase, but is not limited thereto.

[0046] A third embodiment of the present invention relates to a kit for multi-diagnostic of viruses in Solanaceae crops, comprising a primer set for multi-diagnostic of viruses in Solanaceae crops.

[0047] In addition to the marker composition for multiple diagnosis of Solanaceae crop viruses described above, the kit may additionally include reagents generally used in methods for detecting nucleic acids. For example, it may include metal ion salts such as dNTPs (deoxynulceotide triphosphate), heat-resistant polymerase, and magnesium chloride required for PCR reactions, and may include dNTPs and sequenase required for sequencing, but is not limited thereto.

[0048] The above kit may be in the form of a bottle, tub, sachet, envelope, tube, ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc.

[0049] The container of the above kit may be fitted with a cap that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means and is fully or partially detachable. The container may also be fitted with a stopper that allows access to the contents by a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0050] A fourth embodiment of the present invention provides a method for multiplex diagnosis of viruses in Solanaceae crops, comprising: (a) a step of extracting RNA from an infected plant infected with a virus; (b) a step of performing an amplification reaction using the RNA extracted in step (a) as a template and the marker composition of claim 11; and (c) a step of detecting the amplification product obtained in step (b).

[0051] The amplification in step (b) above is preferably performed using the multiplex qRT-PCR method, but is not limited thereto.

[0052] The detection of the amplified product in step (c) above may be performed using any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof, but is not limited thereto.

[0053] According to the method for multiple diagnosis of Solanaceae crop viruses using the primer set for multiple diagnosis of Solanaceae crop viruses of the present invention, there is an advantage of being able to diagnose viruses observed in different types of crops within the Solanaceae family early and multiple times.

[0054] The present invention will be described in detail below through examples. However, these examples are merely for the purpose of specifically explaining the present invention, and the scope of the present invention is not limited by these examples.

[0056] <Preparation Example 1> Securing RNA base sequences of 9 types of Solanaceae crop viruses

[0057] To develop a virus detection kit for Solanaceae crops as hosts, the nucleotide sequence of the virus was obtained. The nucleotide sequence of the target gene was confirmed using the NCBI nucleotide BLAST tool with the nucleotide sequence of the primer set proposed in the notice.

[0058] The obtained nucleotide sequences were analyzed using the NCBI nucleotide BLAST tool for 100 strains of broad bean wilt virus (BBWV), 24 strains of beet western yellow virus (BWYV), 62 strains of tomato bushy stunt virus (TBSV), 57 strains of tomato chlorosis virus (Tomato Yellow Virus), 49 strains of pepper mottle virus (PepMoV), 100 strains of pepper mild mottle virus (PMMoV), 100 strains of cucumber mosaic virus (CMV), 100 strains of tomato spotted wilt virus (TSWV), and tomato yellow leaf curl virus. 100 strains were obtained for each TYLCV.

[0059] To design a set of primers and probes to detect nine viruses, each gene region was selected. BBWV (NCBI accession No: MH447988) polyprotein gene (Sequence No. 28), BWYV (NCBI accession No: AF473561.1) ORF (Open reading frame) gene (Sequence No. 29), CMV (NCBI accession No: LC528236.1) coating protein gene (Sequence No. 30), PepMoV (NCBI accession No: KX446795.1) coating protein gene (Sequence No. 31), PMMoV (NCBI accession No: MN057681.1) coating protein gene (Sequence No. 32), TBSV (NCBI accession No: GQ206144.1) RNA polymerase gene (Sequence No. 33), ToCV (NCBI accession No: EU625350.1) RNA2 protein gene (Sequence No. 34), TSWV (NCBI accession No: The AJ418781.1) nucleocapsid protein gene (sequence number 35) and YLCV (NCBI accession No: MN966555.1) V1 protein gene (sequence number 36) regions were selected (Fig. 2).

[0061] <Preparation Example 2> Securing Synthetic Genes of 9 Solanaceae Crops Viruses

[0062] To obtain RNA from 9 types of Solanaceae crop viruses, the gene regions of each virus were synthesized into DNA. To proceed with transcription from the synthesized DNA into RNA, a T7 procoter was inserted in front of the gene regions to synthesize plasmid DNA.

[0063] RNA was synthesized using the Megascript™ T7 Transcription Kit (Invitrogen) with the synthesized plasmid DNA. The concentrations of the synthesized plasmid DNA and RNA were quantified using a Qubit Flex Fluorometer (Thermo Fisher Scientific) before use.

[0065] <Preparation Example 3> Design of Primer and Probe Sets for 9 Solanaceae Crop Viruses

[0066] To design primer sets for each virus, the Primer3 program was used to design PCR products of 100-200 bp, and under design conditions, the GC content was 20-70%, the length was 17-30 bp, and the melting temperature (Tm) was 55-65°C.

[0067] To design the probe, the qPCR Primer & Probe Design Tool from Eurofins Genomics was referenced, and the primer set was designed to achieve the criteria of a nucleotide oligo length of 25–32 bp, a melting temperature (Tm) of 65–69°C, and a GC ratio of 40–70% within the designed region.

[0068] The list of designed primer sets and probes is summarized in Tables 1 and 2 below.

[0069] Candidate primer set for detecting viruses in Solanaceae crops Sequence number name order 19 BBWV2 F_1 ATTYAGYYGTTCTTGGCYTGYG 20 BBWV2 R_1 GCCRTCTCRTTGGCATGGR 21 BBWV2 F_2 GGCTTGTGTGGCTTCGGAAAG 22 BBWV2 R_2 CAGYAYGATGCAAATGGCCGTGG 23 BBWV2 F_3 AAAACAAACAGCTTTCGTTCCGA 24 BBWV2 R_3 GCCATCTCATTGGCATGGA 25 BWYV F_1 CGCCAACGTCACCGTTTG 26 BWYV F_2 GCCTCATTACATTCCTCAGGCG 27 BWYV R_1 GCTCAGCATCGGCACACC 28 CMV F_1 CCGTAAAGTTCCTGCCTCATCGG 29 CMV R_1 CATGAAGTACCAATTCGTCCGTCTCG 30 CMV F_2 CTACCGTGTGGGTGACAGTC 31 CMV R_2 CGGCGTACTTTCTCATGTCGC 32 CMV F_3 GGGTGACAGTCCGTAAAGTTCC 33 CMV R_3 GATGTAGGAATGCGTTGGTGC 34 PepMoV F_1 GTCACATCGCGTACGTCAACAC 35 PepMoV R_1 GGCTCACATCTACAGTGGTGTG 36 PMMoV F_1 CCTACAACTGCCGAGACGCTTG 37 PMMoV R_1 GTTGTAGCCCAGGTGAGTCCACTC 38 PMMoV F_2 GGGTTTGAATAAGGAAGGGAAGC 39 PMMoV R_2 GTGTACTTCGGCGTTAGGCAATC 40 PMMoV F_3 CTGCCGAGACGCTTGATG 41 PMMoV R_3 GAGTCCACTCGCGCTCTCG 42 TBSV F_1 CAGGAGGTGCTAGAGGTTCG 43 TBSV R_1 GGCTACCCCAACCTTCTCAC 44 ToCV F_1 GACGGCATTTTGGAACTGTCG 45 ToCV R_1 CGACGGAGTGTTGAACATGG 46 TSWV F_1 CATCTCAAAGCTATCAACTGAAGC 47 TSWV R_1 CAGGGTCAGGCTTGTAGAGG 48 TSWV F_2 GCCAAGACAACACTGATCATCTC 49 TSWV R_2 AGGCTTGTAGGAAACTGGG 50 translation F_1 GTAAGGGCCCGTGACTATGTCG 51 translation F_2 CTATGTCGAAGCGACCAGGC 52 translation R_1 GCTTCCGGTACATGGGCCTG 53 translation R_2 GCCTTCACATCCACGAGGAAC 54 translation R_3 CGTCAGGGCTTCGATACATTCTG

[0070] 가지과 작물 바이러스 검출 프로브 후보 염기서열 이름 서열 55 BBWV2 LNA probe FAM-CCCGGAAAAAGGGAGYGYGAYYAAAAGCG-BHQ1 56 BBWV2 LNA probe FAM-AGGGAGTGTGATTAAAAGCGCACCA-BHQ1 57 BBWV2 set5_TaqMan probe FAM-CCRGARCAYGCYTTRGCRGCWAGYGGRAA-BHQ1 58 BBWV2 NTP1 MGB FAM-CAGCTTTCAGTTACTAAACAGCTTTCGTTC-BHQ1 59 BWYV TaqMan probe HEX-GACTGCCCCCCACCCAAGACGTTGCAG-BHQ1 60 BWYV NTP MGB HEX-CCACCCAAGACGTTGCAGGA-BHQ1 61 CMV TaqMan probe_1 FAM-CCGCCATCTCTGCTATGTTGCGGACGGAG-BHQ1 62 CMV TaqMan probe_2 FAM-CGTCGTGGTTCCCGCTCCGCTTCC-BHQ1 63 PepMoV LNA probe HEX-CGAAGCCCATATCCAAATGAAAGCGGCGGC-BHQ1 64 PepMoV TaqMan probe HEX-CCCATATCCAAATGAAAGCGGGCGGCATTG-BHQ1 65 PepMoV TaqMan probe_2 HEX-CGAGGCGGGTAGATGATGCGACGGTGGC-BHQ1 66 PMMoV LNA probe_1 Texas Red-CGAAGCCCATATCCAAATGAAAGCAGCAGCC-BHQ1 67 PMMoV TaqMan probe_2 Texas Red-GGCAGAACTCGGAGTCATCGGACGCCG-BHQ1 68 PMMoV TaqMan probe_3 Texas Red-CGAGGCGGGTAGATGATGCGACGGTGGC-BHQ1 69 TBSV TaqMan probe Texas Red-GGTTGCAGGTTGCCCATCACAGGCAGGG-BHQ1 70 ToCV TaqMan probe FAM-CCACTGGCAAGTCGTGGGTCCTTCCGAAC-BHQ1 71 TSWV TaqMan probe HEX-CCTCCCAGCATTATGGCAAGCCTCACAGAC-BHQ1 72 tylcv TaqMan probe Texas Red-CGCCCGTCTCGAAGGTTCGCCCGAAGGC-BHQ1 73 tylcv TaqMan probe_2 Texas Red-CCACGCCCGTCTCGAAGGTTCGCCG-BHQ1

[0071] <Example 1> Confirmation of Optimal Primer Set and Probe for Detection of 9 Solanaceae Crop Viruses 1. Selection of detection primers for 9 types of Solanaceae crop viruses

[0072] For primer selection, synthesized plasmid DNA (positive control) 10 7 It was used after dilution to copies / µl. Real-time PCR was performed for primer selection, and the conditions and methods are as follows.

[0073] PCR protocol step temperature hour Cycle Free denaturation 95℃ 10 min 1 denaturalization 95℃ 5 sec 40 Annealing & Synthesis 58℃ 30 sec**

[0074] ※ SyBr green fluorescence measurement during the annealing and extension process. To perform real-time PCR, 1 µl of forward primer (10 pmole), 1 µl of reverse primer (1 pmole), 1 µl of SyBr Green (Biofact), and EzAmp ™ A PCR composition with a total volume of 20 µl was prepared using 10 µl of HS One-Step RT-qPCR 2X Master Mix (Elpis biotech), 6 µl of DW, and 1 µl of DNA (>1 ng) and the experiment was conducted.

[0075] For the primers used in the real-time PCR composition, a screening process was conducted on a total of 38 primers for 9 viruses. One pair of primer sets with the best PCR efficiency was selected for each virus, based on the amplification curve results showing a Ct value less than 20, no false positive results in NTC, and reproducibility in two-repetition results (Figs. 1a and 1b).

[0076] The list of final selected primers, as shown in Figures 1a and 1b, is as shown in Table 4 below.

[0077] Final Screening List of Primer Sets for Solanaceae Crop Virus Detection Sequence number name order 1 BBWV2_F3 AAAACAAACAGCTTTCGTTCCGA 2 BBWV2 R_3 GCCATCTCATTGGCATGGA 3 BWYV F_2 GCCTCATTACATTCCTCAGGCG 4 BWYV R_1 GGTGTGCCGATGCTGAGC 5 TBSV F_1 CAGGAGGTGCTAGAGGTTCG 6 TBSV R_1 GTGAGAAGGTTGGGGTAGCC 7 ToCV F_1 GCGTCGGTGGTAATGCTCTGTTC 8 ToCV R_1 GTCAATGCGTTTACGGTCCGGA 9 PepMoV F_1 GTCACATCGCGTACGTCAACAC 10 PepMoV R_1 CACACCACTGTAGATGTGAGCC 11 PMMoV F_3 CTGCCGAGACGCTTGATG 12 PMMoV R_3 CGAGAGCGCGAGTGGACTC 13 CMV F_1 CCGTAAAGTTCCTGCCTCATCGG 14 CMV R_3 GCACCAACGCATTCCTACATC 15 TSWV F_2 GCCAAGACAACACTGATCATCTC 16 TSWV R_2 CCCAGTTTCCTCTACAAGCCT 17 TYLCV_F1 GTAAGGGCCCGTGACTATGTCG 18 TYLCV_R2 GTTCCTCGTGGATGTGAAGGC

[0078] 1-2. Selection of detection probes for 9 types of Solanaceae crop viruses. RT-qPCR was performed using ssRNA diluted to 1 ng / ul as a template to select probes using raw materials whose validity was confirmed via real-time PCR. qRT-PCR was performed to select nucleotide probes, and the conditions and methods are as shown in Table 5 below.

[0079] PCR protocol step temperature hour cycle Reverse Warrior 50℃ 30 min 1 Free denaturation 95℃ 10 min 1 denaturalization 95℃ 5 sec 40 Annealing & Synthesis 58℃ 30 sec**

[0080] ※ To perform qRT-PCR with fluorescence (FAM, HEX, Texas Red) measurement during the annealing and extension process, a PCR composition with a total volume of 20 µl was prepared using 1 µl of forward primer (10 pmole), 1 µl of reverse primer (10 pmole), 1 µl of probe (10 pmole), 10 µl of EzAmp™ HS One-Step RT-qPCR 2X Master Mix (Elpis biotech), 6 µl of DW, and 1 µl of RNA (1 ng), and the experiment was conducted.

[0081] The primers used in the qRT-PCR composition were the 9 pairs of primers finally selected in “1-1. Primer Selection,” and a total of 19 candidate nucleotide probes were applied to confirm the detection results.

[0082] In the experimental results, one probe was selected for each virus that showed a Ct value less than 20 in the amplification curve results, did not produce false positive results in NTC, and had the best PCR efficiency. The final list of probes is shown in Table 6 below, and the selection results are shown in Figures 3a and 3b.

[0083] Sequence number name order 19 BBWV2 NTP1 MGB FAM-CAGCTTTCAGTTACTAAACAGCTTTCGTTC-BHQ1 20 BWYV NTP MGB HEX-CCACCCAAGACGTTGCAGGA-BHQ1 21 TBSV TaqMan probe Texas Red-GGTTGCAGGTTGCCCATCACAGGCAGGG-BHQ1 22 ToCV TaqMan probe FAM-CCACTGGCAAGTCGTGGGTCCTTCCGAAC-BHQ1 23 PepMoV TaqMan probe_2 HEX-CGAGGCGGGTAGATGATGCGACGGTGGC-BHQ1 24 PMMoV TaqMan probe_3 Texas Red-CGAGGCGGGTAGATGATGCGACGGTGGC-BHQ1 25 CMV TaqMan probe_1 FAM-CCGCCATCTCTGCTATGTTGCGGACGGAG-BHQ1 26 TSWV TaqMan probe HEX-CCTCCCAGCATTATGGCAAGCCTCACAGAC-BHQ1 27 TYLCV TaqMan probe_2 Texas Red-CCACGCCCGTCTCGAAGGTTCGCCG-BHQ1

[0084] <Example 2> Establishment of Multiplex qRT-PCR Conditions for 9 Solanaceae Crop Viruses Multiplex qRT-PCR experiments were performed for three virus groups to establish three multiplex qRT-PCR diagnostic methods using selected primer pairs and probes.

[0085] For virus grouping to perform multiplex in silicoAnalysis was performed. To determine the potential for PCR inhibition when multiplexing was conducted with the selected primers and probes, self-dimer, hairpin, and heterodimer phenomena of the primers were predicted, and after examining the potential for PCR in non-target sequences for target-specific reactions, groups #A, #B, and #C were selected for multiplexing (Figs. 4, 5).

[0086] To confirm the detection performance of each virus using a multiplex qRT-PCR diagnostic method with selected primer sets and probes, RT-qPCR was performed using ssRNA diluted to 1 ng / µl as a template, and the method and composition are as shown in Tables 7 to 9 below.

[0087] The procedure was carried out in the same manner as in Table 6 above, and for the multiplex composition, a multiplex qRT-PCR composition was prepared using 3 primer pairs and 3 probes per set (Tables 7 to 9 below).

[0088]

[0089]

[0090]

[0091] For the multiplex qRT-PCR detection method in tube #A, a PCR composition with a total volume of 20 µl was prepared using BBWV2_F3 10 pmole 1 µl, BBWV2 R_3 10 pmole 1 µl, BWYV F_2 10 pmole 1 µl, BWYV R_1 10 pmole 1 µl, TBSV F_1 10 pmole 1 µl, TBSV R_1 10 pmole 1 µl, BBWV2 NTP1 MGB 10 pmole 1 µl, BWYV NTP MGB 10 pmole 0.5 µl, TBSV TaqMan probe 10 pmole 1 µl, EzAmp™ HS One-Step RT-qPCR 2X Master Mix 2X 10 µl, DW 0.5 µl, and RNA 1 ng 1 µl, and the experiment was conducted.

[0092] For the multiplex qRT-PCR detection method in tube #B, a PCR composition of a total volume of 20 µl was prepared using ToCVF_1 10 pmole 1 µl, ToCV R_1 10 pmole 1 µl, PepMoVF_1 10 pmole 1 µl, PepMoV R_1 10 pmole 1 µl, PMMoV F_3 10 pmole 1 µl, PMMoVR_3 10 pmole 1 µl, ToCVTaqMan probe 10 pmole 0.5 µl, PepMoVTaqMan probe_2 10 pmole 1 µl, PMMoVTaqMan probe_3 10 pmole 0.5 µl, EzAmp™ HS One-Step RT-qPCR 2X Master Mix 2X 10 µl, DW 1 µl, and RNA 1 ng 1 µl, and the experiment was conducted.

[0093] For the multiplex qRT-PCR detection method in tube #C, a PCR composition with a total volume of 20 µl was prepared using CMV F_1 10 pmole 1 µl, CMV R_3 10 pmole 1 µl, TSWV F_2 10 pmole 1 µl, TSWV R_2 10 pmole 1 µl, TYLCV_F1 10 pmole 1 µl, TYLCV_R2 10 pmole 1 µl, CMV TaqMan probe_1 10 pmole 0.5 µl, TSWV TaqMan probe 10 pmole 1 µl, TYLCV TaqMan probe_2 10 pmole 0.5 µl, EzAmp™ HS One-Step RT-qPCR 2X Master Mix 2X 10 µl, DW 1 µl, and RNA 1 ng 1 µl, and the experiment was conducted.

[0094] The primers used in the qRT-PCR composition were the 9 pairs of primers finally selected in “1-1. Primer Selection,” and the detection results were confirmed by applying a total of 9 candidate nucleotide probes.

[0095] In the experimental results, the detection result was determined based on whether the Ct value in the amplification curve result was less than 20 and whether a false positive result occurred in the NTC, and the result is as shown in Fig. 6.

[0097] <Example 3> Sensitivity analysis of the primer set and probe of the present invention

[0098] To investigate the diagnostic sensitivity of the primer set and probe of the present invention, RNA of 9 types of Solanaceae crop viruses was diluted from 10 ng to 100 fg, and the Ct values ​​of the amplification curve results were analyzed, confirming that the primer set and probe of the present invention can diagnose synthetic RNA of 9 types of viruses from 1 fg to 10 fg (Figs. 7 to 9).

[0099] It was found that it is possible to screen nine types of Solanaceae crop viruses at once in a single experiment, and that the detection results are excellent. The present invention has the advantage of being able to distinguish and diagnose nine types of viruses while saving time and cost compared to conventional simple screening.

[0101] As specific parts of the content of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention.

[0102] Accordingly, the substantial scope of the present invention shall be defined by the appended claims and their equivalents. Simple variations or modifications of the present invention may be readily utilized by those skilled in the art, and all such variations or modifications shall be deemed to be included within the scope of the present invention.

Claims

Claim 1 A first primer set for diagnosing cucumber mosaic virus (CMV) comprising a forward primer of SEQ ID NO. 13, a reverse primer pair of SEQ ID NO. 14, and a probe of SEQ ID NO. 25; a second primer set for diagnosing tomato spotted wilt virus (TSWV) comprising a forward primer of SEQ ID NO. 15, a reverse primer pair of SEQ ID NO. 16, and a probe of SEQ ID NO. 26; A primer set for multiplex diagnosis of viruses in Solanaceae crops, comprising a third primer set for diagnosing Tomato Yellow Leaf Curl Virus (TYLCV) consisting of a forward primer of SEQ ID NO. 17, a reverse primer pair of SEQ ID NO. 18, and a probe of SEQ ID NO. 27, wherein the multiplex diagnosis primer set is capable of diagnosing up to 10 fg of synthetic RNA of Cucumber Mosaic Virus (CMV), Tomato Spotted Wilt Virus (TSWV), and Tomato Yellow Leaf Curl Virus (TYLCV), and wherein the Solanaceae crop is one or more selected from paprika, tomato, eggplant, potato, and bell pepper. Claim 2 delete Claim 3 A marker composition for multiplex diagnosis of viruses in Solanaceae crops comprising a primer set for multiplex diagnosis of viruses in Solanaceae crops according to claim 1, wherein the Solanaceae crop is one or more selected from paprika, tomato, eggplant, potato, and bell pepper. Claim 4 A kit for multi-diagnostic of viruses in Solanaceae crops comprising a marker composition for multi-diagnostic of viruses in Solanaceae crops according to Paragraph 3, wherein the Solanaceae crop is one or more selected from paprika, tomato, eggplant, potato, and bell pepper. Claim 5 A method for multiplex diagnosis of viruses in Solanaceae crops, comprising: (a) a step of extracting RNA from a virus-infected plant; (b) a step of performing an amplification reaction using the RNA extracted in step (a) as a template and the marker composition of claim 3; and (c) a step of detecting the amplification product obtained in step (b), wherein the Solanaceae crop is one or more selected from paprika, tomato, eggplant, potato, and bell pepper. Claim 6 A multiplex diagnostic method for viruses in Solanaceae crops, characterized in that, in claim 5, the amplification in step (b) above utilizes a multiplex quantitative polymerase chain reaction (multiplex qRT-PCR) method. Claim 7 A method for multiplex diagnosis of viruses in Solanaceae crops according to claim 5, wherein the detection of the amplification product of step (c) above is performed using any one method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, phosphorescence measurement, and combinations thereof.

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